Antibodies to human programmed death receptor pd-1

ABSTRACT

Antibodies which block the binding of human Programmed Death Receptor 1(hPD-1) to its ligands (hPD-L1 or hPD-L2) and their variable region sequences are disclosed. A method of increasing the activity (or reducing downmodulation) of an immune response through the PD-I pathway is also disclosed.

BACKGROUND OF THE INVENTION

Programmed death receptor 1 (PD-1) is an immunoinhibitory receptor thatis primarily expressed on activated T and B cells. Interaction with itsligands has been shown to attenuate T-cell responses both in vitro andin vivo. Blockade of the interaction between PD-1 and one of itsligands, PD-L1, has been shown to enhance tumor-specific CD8⁺ T-cellimmunity and may therefore be helpful in clearance of tumor cells by theimmune system.

PD-1 (encoded by the gene Pdcd1) is an Immunoglobulin superfamily memberrelated to CD28, and CTLA-4. PD-1 has been shown to negatively regulateantigen receptor signaling upon engagement of its ligands (PD-L1 and/orPD-L2) The structure of murine PD-1 has been solved as well as theco-crystal structure of mouse PD-1 with human PD-L1 (Zhang, X. et al.,Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105:3011-6 (2008)). PD-1 and like family members are type I transmembraneglycoproteins containing an Ig Variable-type (V-type) domain responsiblefor ligand binding and a cytoplasmic tail that is responsible for thebinding of signaling molecules. The cytoplasmic tail of PD-1 containstwo tyrosine-based signaling motifs, an ITIM (immunoreceptortyrosine-based inhibition motif) and an ITSM (immunoreceptortyrosine-based switch motif).

Following T cell stimulation, PD-1 recruits the tyrosine phosphataseSHP-2 to the ITSM motif within its cytoplasmic tail, leading to thedephosphorylation of effector molecules such as CD3 zeta, PKC theta andZAP70 that are involved in the CD3 T cell signaling cascade. Themechanism by which PD-1 downmodulates T cell responses is similar to,but distinct from that of CTLA-4, as both molecules regulate anoverlapping set of signaling proteins (Parry et al., Mol. Cell. Biol.25: 9543-9553.). Bennett and coworkers have shown that PD-1-mediatedinhibition of T-cell signaling is only effective when both activatingand inhibitory signals are on the same surface, indicating that the PD-1signaling mechanism is spatiotemporally determined (Bennett F. et al., JImmunol. 170:711-8 (2003)).

PD-1 was shown to be expressed on activated lymphocytes (peripheral CD4⁺and CD8⁺ T cells, B cells and monocytes) and has also been shown to beexpressed during thymic development on CD4⁻CD8⁻ (double negative) Tcells as well as NK-T cells.

The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed orcan be induced in a variety of cell types, including non-hematopoietictissues as well as various tumor types. PD-L1 is expressed on B, T,myeloid and dendritic cells (DCs), but also on peripheral cells, likemicrovascular endothelial cells and non-lymphoid organs like heart, lungetc. In contrast, PD-L2 is only found on macrophages and DCs. Theexpression pattern of PD-1 ligands is suggestive of a role for PD-1 inmaintaining peripheral tolerance and may serve to regulate self-reactiveT- and B-cell responses in the periphery. Both ligands are type Itransmembrane receptors containing both IgV- and IgC-like domains in theextracellular region. Both ligands contain short cytoplasmic regionswith no known signaling motifs.

To date, numerous studies have shown that interaction of PD-1 with itsligands leads to the inhibition of lymphocyte proliferation in vitro andin vivo. Disruption of the PD-1/PD-L1 interaction has been shown toincrease T cell proliferation and cytokine production and blockprogression of the cell cycle. Initial analysis of Pdcd1^(−/−) mice didnot identify any drastic immunological phenotype. However aged micedeveloped spontaneous autoimmune diseases which differ according to thestrain onto which the Pdcd1 deficiency was backcrossed. These includelupus-like proliferative arthritis (C57BL/6) (Nishimura H. et al., Int.Immunol. 10: 1563-1572 (1998)), fatal cardiomyopathy (BALB/c) (NishimuraH. et al., Science 291: 319-322 (2001)) and type I diabetes (NOD) (WangJ. et al., Proc. Natl. Acad. Sci. U.S.A 102: 11823-11828 (2005)).Overall, analysis of the knockout animals has led to the understandingthat PD-1 functions mainly in inducing and regulating peripheraltolerance. Thus, therapeutic blockade of the PD-1 pathway may be helpfulin overcoming immune tolerance. Such selective blockade may be of use inthe treatment of cancer or infection as well as in boosting immunityduring vaccination (either prophylactic or therapeutic).

The role of PD-1 in cancer is established in the literature. It is knownthat tumor microenvironment can protect tumor cells from efficientimmune destruction. PD-L1 has recently been shown to be expressed on anumber of mouse and human tumors (and is inducible by IFN gamma on themajority of PD-L1 negative tumor cell lines) and is postulated tomediate immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A.99: 12293-12297 (2002); Strome S. E. et al., Cancer Res., 63: 6501-6505(2003).

In humans, expression of PD-1 (on tumor infiltrating lymphocytes) and/orPD-L1 (on tumor cells) has been found in a number of primary tumorbiopsies assessed by immunohistochemistry. Such tissues include cancersof the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast,kidney, esophagus, stomach, oral squamous cell, urothelial cell, andpancreas as well as tumors of the head and neck (Brown J. A. et al., J.Immunol. 170: 1257-1266 (2003); Dong H. et al., Nat. Med. 8: 793-800(2002); Wintterle et al., Cancer Res. 63: 7462-7467 (2003); Strome S. E.et al., Cancer Res., 63: 6501-6505 (2003); Thompson R. H. et al., CancerRes. 66: 3381-5 (2006); Thompson et al., Clin. Cancer Res. 13: 1757-61(2007); Nomi T. et al., Clin. Cancer Res. 13: 2151-7. (2007)). Morestrikingly, PD-ligand expression on tumor cells has been correlated topoor prognosis of cancer patients across multiple tumor types (reviewedin Okazaki and Honjo, Int. Immunol. 19: 813-824 (2007)).

Blockade of the PD-1/PD-L1 interaction could lead to enhancedtumor-specific T-cell immunity and therefore be helpful in clearance oftumor cells by the immune system. To address this issue, a number ofstudies were performed. In a murine model of aggressive pancreaticcancer, T. Nomi et al. (Clin. Cancer Res. 13: 2151-2157 (2007))demonstrated the therapeutic efficacy of PD-1/PD-L1 blockade.Administration of either PD-1 or PD-L1 directed antibody significantlyinhibited tumor growth. Antibody blockade effectively promoted tumorreactive CD8⁺ T cell infiltration into the tumor resulting in theup-regulation of anti-tumor effectors including IFN gamma, granzyme Band perforin. Additionally, the authors showed that PD-1 blockade can beeffectively combined with chemotherapy to yield a synergistic effect. Inanother study, using a model of squamous cell carcinoma in mice,antibody blockade of PD-1 or PD-L1 significantly inhibited tumor growth(Tsushima F. et al., Oral Oncol. 42: 268-274 (2006)).

In other studies, transfection of a murine mastocytoma line with PD-L1led to decreased lysis of the tumor cells when co-cultured with atumor-specific CTL clone. Lysis was restored when anti-PD-L1 mAb wasadded (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99: 12293-12297(2002)). In vivo, blocking the PD1/PD-L1 interaction was shown toincrease the efficacy of adoptive T cell transfer therapy in a mousetumor model (Strome S. E. et al., Cancer Res. 63: 6501-6505 (2003)).Further evidence for the role of PD-1 in cancer treatment comes fromexperiments performed with PD-1 knockout mice. PD-L1 expressing myelomacells grew only in wild-type animals (resulting in tumor growth andassociated animal death), but not in PD-1 deficient mice (Iwai Y. etal., Proc. Natl. Acad. Sci. U.S.A. 99: 12293-12297 (2002)).

In human studies, R. M. Wong et al. (Int. Immunol. 19: 1223-1234 (2007))showed that PD-1 blockade using a fully human anti-PD-1 antibodyaugmented the absolute numbers of tumor-specific CD8+ T cells (CTLs) inex vivo stimulation assays using vaccine antigens and cells fromvaccinated individuals. In a similar study, antibody blockade of PD-L1resulted in enhanced cytolytic activity of tumor-associatedantigen-specific cytotoxic T cells and increased cytokine production bytumor specific T_(H) cells (Blank C. et al., Int. J. Cancer 119: 317-327(2006)). The same authors showed that PD-L1 blockade augmentstumor-specific T cell responses in vitro when used in combination withanti-CTLA-4 blockade.

Overall, the PD-1/PD-L1 pathway is a well-validated target for thedevelopment of antibody therapeutics for cancer treatment. Anti-PD-1antibodies may also be useful in chronic viral infection. Memory CD8⁺ Tcells generated after an acute viral infection are highly functional andconstitute an important component of protective immunity. In contrast,chronic infections are often characterized by varying degrees offunctional impairment (exhaustion) of virus-specific T-cell responses,and this defect is a principal reason for the inability of the host toeliminate the persisting pathogen. Although functional effector T cellsare initially generated during the early stages of infection, theygradually lose function during the course of a chronic infection. Barberet al. (Barber et al., Nature 439: 682-687 (2006)) showed that miceinfected with a laboratory strain of LCMV developed chronic infectionresulting in high levels of virus in the blood and other tissues. Thesemice initially developed a robust T cell response, but eventuallysuccumbed to the infection upon T cell exhaustion. The authors foundthat the decline in number and function of the effector T cells inchronically infected mice could be reversed by injecting an antibodythat blocked the interaction between PD-1 and PD-L1.

Recently, it has been shown that PD-1 is highly expressed on T cellsfrom HIV infected individuals and that receptor expression correlateswith impaired T cell function and disease progression (Day et al.,Nature 443:350-4 (2006).; Trautmann L. et al., Nat. Med. 12: 1198-202(2006)). In both studies, blockade of the ligand PD-L1 significantlyincreased the expansion of HIV-specific, IFN-gamma producing cells invitro.

Other studies also implicate the importance of the PD-1 pathway incontrolling viral infection. PD-1 knockout mice exhibit better controlof adenovirus infection than wild-type mice (Iwai et al., J. Exp. Med.198:39-50 (2003)). Also, adoptive transfer of HBV-specific T cells intoHBV transgenic animals initiated hepatitis (Isogawa M. et al., Immunity23:53-63 (2005)). The disease state of these animals oscillates as aconsequence of antigen recognition in the liver and PD-1 upregulation byliver cells.

BRIEF SUMMARY OF THE INVENTION

The invention provides isolated antibodies and antibody fragments thatbind to human and cyno PD-1. In some embodiments, the antibody orantibody fragment blocks binding of human PD-L1 and human PD-L2 to humanPD-1. In some embodiments, the PD-1 antibody or antibody fragment of theinvention includes one or more CDRs (antibodyComplementarity-Determining Regions) selected from SEQ ID NOs: 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; and in further embodiments,includes one or more heavy chain CDRs of SEQ ID NOs:12, 13, 14, 18, 19and 20 and/or the light chain CDRs of SEQ ID NOs: 9, 10, 11, 15, 16 and17. In some embodiments, the antibody or antibody fragment is a chimericantibody, human antibody, humanized antibody or a fragment thereof.

In one embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a light chaincomprising CDRs SEQ ID NOs: 9, 10 and 11, or variants of any saidsequences; and/or a heavy chain comprising CDRs SEQ ID NOs: 12, 13 and14, or variants of any said sequences.

In another embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a light chaincomprising CDRs SEQ ID NOs: 15, 16 and 17 or variants of any saidsequences; and/or a heavy chain comprising CDRs SEQ ID NOs: 18, 19 and20, or variants of any said sequences.

In one embodiment, the invention comprises an antibody or antigenbinding fragment comprising a heavy chain variable region SEQ ID NO: 5or a variant thereof; and/or a light chain variable region comprisingSEQ ID NO: 6 or a variant thereof.

In one embodiment, the invention comprises an antibody or antigenbinding fragment comprising a heavy chain variable region SEQ ID NO: 7or a variant thereof and/or a light chain variable region comprising SEQID NO: 8 or a variant thereof.

In one embodiment, the invention comprises an antibody or antigenbinding fragment comprising a heavy chain variable region comprisingamino acid residues 20 to 139 of SEQ ID NO: 30 or a variant thereof;and/or a light chain variable region comprising comprising amino acidresidues 20 to 130 of SEQ ID NO: 32 or a variant thereof.

In one embodiment, the invention comprises an antibody or antigenbinding fragment comprising a heavy chain variable region comprisingamino acid residues 20 to 139 of SEQ ID NO: 30 or a variant thereof;and/or a light chain variable region comprising comprising amino acidresidues 20 to 130 of SEQ ID NO: 33 or a variant thereof.

In one embodiment, the invention comprises an antibody or antigenbinding fragment comprising a heavy chain variable region comprisingamino acid residues 20 to 139 of SEQ ID NO: 30 or a variant thereof;and/or a light chain variable region comprising comprising amino acidresidues 20 to 130 of SEQ ID NO: 34 or a variant thereof.

In one embodiment, the invention comprises an antibody or antigenbinding fragment comprising a heavy chain variable region comprising anamino acid sequence having at least 90% homology to amino acid residues20 to 139 of SEQ ID NO: 30; and/or a light chain variable regioncomprising and an amino acid sequence having at least 90% homology toamino acid residues 20 to 130 of SEQ ID NO: 32, 33 or 34.

In one embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a heavy chaincomprising amino acid residues 20 to 466 of SEQ ID NO: 31 or a variantthereof, and/or a light chain comprising amino acid residues 20 to 237of SEQ ID NO: 36 or a variant thereof.

In one embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a heavy chaincomprising the amino acid residues 20 to 466 of SEQ ID NO: 31 or avariant thereof, and/or a light chain comprising the amino acid residues20 to 237 of SEQ ID NO: 37 or a variant thereof.

In one embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a heavy chaincomprising amino acid residues 20 to 466 of SEQ ID NO: 31 or a variantthereof, and/or a light chain comprising amino acid residues 20 to 237of SEQ ID NO: 38 or a variant thereof.

In one embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a heavy chaincomprising amino acid residues 20 to 469 of SEQ ID NO: 35 or a variantthereof, and/or a light chain comprising amino acid residues 20 to 237of SEQ ID NO: 36 or a variant thereof.

In one embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a heavy chaincomprising amino acid residues 20 to 469 of SEQ ID NO: 35 or a variantthereof, and/or a light chain comprising amino acid residues 20 to 237of SEQ ID NO: 37 or a variant thereof.

In one embodiment, the invention provides an isolated antibody orantibody fragment which binds to human PD-1 comprising: a heavy chaincomprising amino acid residues 20 to 469 of SEQ ID NO: 35 or a variantthereof, and/or a light chain comprising amino acid residues 20 to 237of SEQ ID NO: 38 or a variant thereof.

In any of the above embodiments, the variant of the antibody or antibodyfragment fragment of the invention may comprise one, two or threeconservatively modified amino acid substitutions.

In any of the above embodiments, the antibody or antibody fragment ofthe invention may comprise a human heavy chain constant region or avariant thereof, wherein the variant comprises up to 20 conservativelymodified amino acid substitutions; and/or a human light chain constantregion or a variant thereof, wherein the variant comprises up to 20conservatively modified amino acid substitutions. In some embodiments,the variant may comprise up to 10 conservatively modified amino acidsubstitutions. In some embodiments, the variant may comprise up to 5conservatively modified amino acid substitutions. In some embodiments,the variant may comprise up to 3 conservatively modified amino acidsubstitutions. In any of the above embodiments, the human heavy chainconstant region or variant thereof may be of the IgG1 or IgG4 isotype.

In any of the above described embodiments, the antibody or antibodyfragment of the invention may bind human PD-1 with a K_(D) of about 100pM or lower. In another embodiment, the antibody or antibody fragmentmay bind human PD-1 with a K_(D) of about 30 pM or lower. In anotherembodiment, the antibody or antibody fragment may bind human PD-1 withabout the same K_(D) as an antibody having a heavy chain comprising theamino acid sequence of SEQ ID NO: 31 and a light chain comprising theamino acid sequence of SEQ ID NO: 32. In another embodiment, theantibody or antibody fragment may bind human PD-1 with about the sameK_(D) as an antibody having a heavy chain comprising the amino acidsequence of SEQ ID NO: 31 and a light chain comprising the amino acidsequence of SEQ ID NO: 33.

In any of the above described embodiments, the antibody or antibodyfragment of the invention may bind human PD-1 with a k_(assoc) of about7.5×10⁵ l/M·s or faster. In one embodiment, the antibody or antibodyfragment may bind human PD-1 with a k_(assoc) of about 1×10⁶ l/M·s orfaster.

In any of the above described embodiments, the antibody or antibodyfragment may bind human PD-1 with a k_(dissoc) of about 2×10⁻⁵ l/s orslower. In one embodiment, the antibody or antibody fragment may bindhuman PD-1 with a k_(dissoc) of about 2.7×10⁻⁵ l/s or slower. In oneembodiment, the antibody or antibody fragment may bind human PD-1 with ak_(assoc) of about 3×10⁻⁵ l/s or slower.

K_(D), k_(assoc) and k_(dissoc) values can be measured using anyavailable method. In preferred embodiments, the disaaociation constantis measured using bio-light interferometry (for example, the ForteBioOctet method described in Example 2). In other preferred embodiments,the disassociation constant can be measured using surface plasmonresonance (e.g. Biacore) or Kinexa.

Further, in any of the above described embodiments, the antibody orantibody fragment of the invention may block binding of human PD-L1 orhuman PD-L2 to human PD-1 with an IC₅₀ of about 1 nM or lower. Theblockade of ligand binding can be measured and the IC₅₀ calculated usingany method known in the art, for example, the FACS or FMAT methodsdescribed in the Examples hereub.

The invention also comprises an antibody or antibody fragment whichcompetes for a binding epitope on human PD-1 with any of the antibodiesdescribed above, and which blocks the binding of human PD-L1 or humanPD-L2 to human PD-1 with an IC₅₀ of about 1 nM or lower.

The invention also comprises an antibody or antibody fragment whichcompetes for a binding epitope on human PD-1 with any of the antibodiesdescribed above, and which binds human PD-1 with a K_(D) of about 100 pMor lower. In one embodiment, the antibody or antibody fragment bindshuman PD-1 with a K_(D) of about 30 pM or lower.

The invention also comprises an antibody or antibody fragment whichcompetes for a binding epitope on human PD-1 with any of the antibodiesdescribed above, and which binds human PD-1 with about the same K_(D) asan antibody having a heavy chain comprising the amino acid sequence ofSEQ ID NO: 31 and a light chain comprising the amino acid sequence ofSEQ ID NO: 32.

The invention also comprises an antibody or antibody fragment thatcompetes for a binding epitope on human PD-1 with any of the antibodiesdescribed above, and which binds human PD-1 with about the same K_(D) asan antibody having a heavy chain comprising the amino acid sequence ofSEQ ID NO: 31 and a light chain comprising the amino acid sequence ofSEQ ID NO: 33.

The invention also comprises an antibody or antibody fragment whichcompetes for a binding epitope on human PD-1 with any of the antibodiesdescribed above, and which binds human PD-1 with a k_(assoc) of about7.5×10⁵ l/M·s or faster. In one embodiment, the antibody or antibodyfragment may bind human PD-1 with a k_(assoc) of about 1×10⁶ l/M·s orfaster.

The invention also comprises an antibody or antibody fragment whichcompetes for a binding epitope on human PD-1 with any of the antibodiesdescribed above, and which binds human PD-1 with a k_(dissoc) of about2×10⁻⁵ l/s or slower. In one embodiment, the antibody or antibodyfragment may bind human PD-1 with a k_(assoc) of about 2.7×10⁻⁵ l/s orslower. In one embodiment, the antibody or antibody fragment may bindhuman PD-1 with a k_(assoc) of about 3×10⁻⁵ l/s or slower.

In some embodiments, the antibody or antibody fragments of the inventionare chimeric antibodies or fragments of chimeric antibodies.

In some embodiments, the antibody or antibody fragments of the inventionare human antibodies or fragments of human antibodies.

In some embodiments, the antibody or antibody fragments of the inventionare humanized antibodies or fragments of humanized antibodies.

In some embodiments, the antibody fragments of the invention are Fab,Fab′, Fab′-SH, Fv, scFv, or F(ab′)₂ antibody fragments.

In some embodiments, the antibody fragments of the invention arediabodies.

The invention also comprises bispecific antibodies comprising any one ofthe antibody or antibody fragments described above that bind to humanPD-1.

In some embodiments, the isolated anti-PD-1 antibodies and antibodyfragments of the invention increase T cell activation as measured bytypical means known to one skilled in the art (including, withoutlimitation, increased immune cell proliferation, increased cytokinesecretion or expression of activation markers such as CD25 and/or CD69).

In any of the above described embodiments, the antibody or antibodyfragment of the invention may enhance the immune response afterstimulation with Staphylococcus Enterotoxin B or Tetanus Toxoid ex vivoor in vivo. The increased immune activation may be determined usingmethods known to anyone skilled in the art, for example, quantifyingproliferation of immune cells (such as T cells) or cytokine productionby immune cells (for example production of IFNγ or IL-2 by T cells).

The invention also comprises nucleic acids encoding the anti-PD-1antibodies and antibody fragments of the invention. Included in theinvention are nucleic acids encoding any one of the amino acid sequencesdisclosed in SEQ ID NOS: 5 to 20 and 30-38 (with or without the leadersequences). Also included withn the invention are nucleic acidscomprising SEQ ID NOS:1 to 4 and 21 to 29 (with or without the nucleicacids encoding the leader sequences).

The invention also comprises cells and expression vectors comprisingnucleic acids encoding the antibodies or antibody fragments of theinvention. Further, the invention comprises a method of producing anantibody or antibody fragment of the invention comprising: (a) culturingthe host cell comprising a nucleic acid encoding an antibody or antibodyfragment of the invention in culture medium under conditions wherein thenucleic acid sequence is expressed, thereby producing polypeptidescomprising the light and heavy chain variable regions; and (b)recovering the polypeptides from the host cell or culture medium.

The invention also comprises compositions comprising an antibody orantibody fragment of the invention in combination with apharmaceutically acceptable carrier or diluent.

The invention also comprises a method of increasing the activity of animmune cell, comprising administering to a subject in need thereof atherapeutically effective amount of an antibody or antibody fragment ofthe invention. In one embodiment, the method may be used to treatcancer. In another embodiment, the method may be use to treat aninfection or infectious disease. In yet another embodiment, the methodmay be used as a vaccine adjuvant. In some embodiments, the methodcomprises further administering a second therapeutic agent or treatmentmodality.

In some embodiments, the invention comprises a method of increasing theactivity of an immune cell, comprising administering to a subject inneed thereof a therapeutically effective amount of an antibody orantibody fragment of the invention, and further comprising measuring Tcell activation ex vivo in a sample derived from the subject, wherein anincrease in T cell activity indicates that the treatment should becontinued. In other embodiments, the invention comprises a method ofincreasing the activity of an immune cell, comprising administering to asubject in need thereof a therapeutically effective amount of anantibody or antibody fragment of the invention, and further comprisingmeasuring T cell activation ex vivo in a sample derived from thesubject, wherein an increase in T cell activity predicts the likelihoodthat the treatment will be successful. In one embodiment, the increasein T cell activity is determined by: (i) measuring SEB inducedproduction of one or more cytokines selected from the group consistingof: IL-2, TNFα, IL-17, IFNγ, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13;or (ii) measuring TT induced production of a cytokine selected from thegroup consisting of: IL-2, TNFα, IL-17, IFNγ, GM-CSF, RANTES, IL-6,IL-8, IL-5 and IL-13.

The invention also comprises the use of an anti-PD-1 antibody orantibody fragment of the invention for the preparation of a medicamentto increase immune response.

The invention also comprises the use of an anti-PD-1 antibody orantibody fragment of the invention for the preparation of a medicamentto treat cancer.

The invention also comprises the use of an anti-PD-1 antibody orantibody fragment of the invention as a vaccine adjuvant.

The invention also comprises an immunoconjugate comprising an anti-PD-1antibody or antibody fragment of the invention, linked to a therapeuticagent such as a bacterial toxin or a radiotoxin. Non-limiting examplesof cytotoxic agents include taxol, cytochalasin B, mitomycin, etoposideand vincristine or other antimetabolites, alkylating agents, antibioticsand antimitotics.

The invention also comprises a method of increasing the activity, orreducing the downmodulation, of an immune cell comprising contacting theimmune cell with any one of the antibodies or antibody fragments of theinvention. This method could be used to treat cancer or infectiousdiseases (such as chronic viral infections), or could be used as anadjuvant to a prophylactic or therapeutic vaccine.

The invention also comprises a method of increasing an immune responseto an antigen, comprising contacting an immune cell with an antigen andan anti-PD-1 antibody or an antibody fragment such that an immuneresponse to the antigen is increased or enhanced. This method could beconducted in vivo (in a subject) or ex vivo.

In some embodiments, an anti-PD-1 antibody or antibody fragment may becombined with a second therapeutic agent or treatment modality. In oneembodiment, an anti-PD-1 antibody or antibody fragment may be combinedwith cancer treatments involving the application of recombinantcytokines or secreted immune factors. Non-limiting examples ofcombinations include combining anti-PD-1 antibody with recombinant IL-2or recombinant IFNα2 for the treatment of melanoma or renal cellcarcinoma. Recombinant IL-2 enhances T cell outgrowth in cancerpatients. Recombinant IFNα2 inhibits cancer cell growth but alsoincreases expression of the inhibitory ligands for PD-1 on cancer cells,antigen-presenting cells and other somatic cells in the treatedpatients. Anti-PD-1 can be combined with other cytokines that might beconsidered useful for the treatment of cancer or infectious diseases.

In some embodiments, anti-PD-1 antibodies or antibody fragments can becombined with a vaccine to prevent or treat cancer or infectiousdisease. As a non-limiting example, anti-PD-1 could be combined with aprotein, peptide or DNA vaccine containing one or more antigens whichare relevant to the cancer or infection to be treated, or a vaccinecomprising of dendritic cells pulsed with such a) antigen. Anotherembodiment includes the use of anti-PD-1 with (attenuated) cancer cellor whole virus vaccines. One embodiment involves a combination ofanti-PD-1 therapy with a whole cell cancer vaccine that is engineered tosecrete GM-CSF.

In some embodiments, anti-PD-1 antibodies or antibody fragments can becombined with treatment that is considered to be standard of care incancer or infectious disease. Rationale for such combinations is thatconcurrent increased immune activation by anti-PD-1 will induce orfacilitate initial clinical response to standard of care treatment,induce durable clinical response and long-term immune control ofdisease.

In one embodiment, treatment with anti-PD-1 antibodies or antibodyfragments may be combined with chemotherapy. Chemotherapy usingcytotoxic agents will result in cancer cell death thereby increasingrelease of tumor antigens. Such increased availability of tumor antigenmay result in synergy with anti-PD-1 treatment. A non-limiting exampleis provided by the use of decarbazine or temozolomide for the treatmentof melanoma and gemcitabine for pancreatic cancer.

In one embodiment, treatment with anti-PD-1 antibodies or antibodyfragments may be combined with radiotherapy. Radiotherapy induces cancercell death and increasing availability of tumor antigens forpresentation and activation of immune cells.

In another embodiment, treatment with anti-PD-1 antibodies or antibodyfragments may be combined with surgery to remove cancer cells from asubject.

In other embodiments, anti-PD-1 antibodies or antibody fragments may becombined with therapies which may result in synergy with PD-1 blockadeincluding targeted agents used for hormone deprivation or inhibition ofangiogenesis, or targeting proteins active in tumor cells, all resultingin enhanced tumor cell death and availability of immune stimulatingtumor antigens. In combination with an anti-PD-1 antibody or antibodyfragment, increased T cell activation may result in durable immunecontrol of cancer.

In some embodiments, an anti-PD-1 antibody or antibody fragment may becombined with another therapeutic antibody useful for the treatment ofcancer or infectious disease. A non-limiting example is provided by thecombination of anti-PD-1 with an antibody targeting Her2/neu ortargeting the EGF receptor. In another non-limiting example, ananti-PD-1 antibody or antibody fragment is combined with treatmenttargeting VEGF or its receptors. In another embodiment, an anti-PD-1antibody or antibody fragment is combined with anti-CTLA-4. In yetanother nonlimiting example, anti-PD-1 is combined with an antibody thattargets RSV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the results of experiments demonstrating thatantibodies immobilized from hybridoma supernatants are able to reduceIL-2 secretion by Jurkat E6.2.11 cells stimulated with immobilizedanti-CD3 and soluble anti-CD28.

FIG. 2 shows the results of experiments demonstrating that antibodiesagainst human PD-1 bind to PD-1. FIG. 2A is a graph showing dosedependent binding of anti-PD-1 antibodies to purified PD-1/Fc in aprotein ELISA. FIG. 2B is a graph showing dose dependent binding of antiPD-1 antibodies to PD-1 expressed on the surface of CHO cellstransfected with hPD-1 in a CELISA.

FIG. 3 shows results of FMAT experiments demonstrating that theantibodies against PD-1 compete for binding of PD-L1 and PD-L2 to CHOcells transfected with human PD-1. FIG. 3A is a graph showing dosedependent inhibition of binding of PD-L1 by hPD-1.08A and hPD-1.09A andto a lesser extent by J116. FIG. 3B is a graph showing dose dependentinhibition of PD-L2.

FIG. 4 is a bar graph which shows results of experiments demonstratingthat SEB-stimulated IL-2 production by healthy donor blood cells isenhanced in the presence of anti-PD-1, anti PD-L1 or anti-CTLA-4antibodies. Bars show the average fold increase in IL-2 across donors(±SEM). Numbers inside each bar indicate the number of donorsrepresented. Mouse (m) IgG1 is the isotype control for anti-PD-1.08A(08A), anti-PD-1.09A (09A) and anti-PD-L1. Mouse (m) IgG2a is theisotype control for anti-CTLA-4. Each IL-2 value is compared to its owncontrol to determine the fold change (fold change IL-2 of 4 means 400%increase in IL-2 production when compared to SEB alone). None=SEB alone.

FIG. 5 shows results of experiments demonstrating that anti-PD-1antibodies promote T cell proliferation and cytokine secretion (IL-2 andIFNγ) when stimulated with the recall antigen tetanus toxoid. FIG. 5shows concentration dependent IFNγ secretion.

FIG. 6 is a graph depicting the k_(assoc) and k_(dissoc) rates foranti-PD-1 antibodies as measured by bio-light interferometry. Diagonallines indicate theoretical calculated K_(D) values. The antibodies arelisted at the right by K_(D) in ascending order.

FIG. 7 is a bar graph which shows results of experiments demonstratingthat SEB-stimulated IL-2 production by healthy donor blood cells isincreased in the presence of 25 ug/ml murine (09A) or humanizedanti-PD-1 antibodies (h409A11, h409A16 and h409A17). Bars show theaverage fold increase in IL-2 across three donors (+SEM). Mouse (m) IgG1is the isotype control for anti-PD-1.09A (09A). Human (h) IgG4 is theisotype control for h409A11, h409A16 and h409A17 antibodies. Each IL-2value is compared to its own control to determine the fold change.None=SEB alone.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

-   hPD-1.08A Murine monoclonal anti-hPD-1 antibody-   hPD-1.09A Murine monoclonal anti-hPD-1 antibody-   08A-VH VH isolated from hPD-1.08A hybridoma-   08A-VK VK isolated from hPD-1.08A hybridoma-   09A-VH VH isolated from hPD-1.09A hybridoma-   09A-VK VK isolated from hPD-1.09A hybridoma-   c109A Chimeric IgG1 version of hPD1.09A antibody-   c109A-VH Chimeric heavy chain, consisting of murine 09A-VH fused to    hIgG1 constant region-   c109A-VK Chimeric light chain, consisting of murine 09A-VK fused to    human kappa constant region-   109A-H Humanized IgG1 09A heavy chain sequence with zero back    mutations.-   409A-H Humanized IgG4-09A heavy chain sequence with zero FWR back    mutations-   K09A-L-11 Humanized 09A-kappa sequence with framework originally    having CDR1 length of 11 AAs-   K09A-L-16 Humanized 09A-kappa sequence with framework originally    having CDR1 length of 16 AAs-   K09A-L-17 Humanized 09A-kappa sequence with framework originally    having CDR1 length of 17 AAs-   h409A11 Humanized IgG4 version of 09A antibody comprising a heavy    chain comprising the sequence of 409A-H and a light chain comprising    the sequence of K09A-L-11-   h409A16 Humanized IgG4 version of 09A antibody comprising a heavy    chain comprising the sequence of 409A-H and a light chain comprising    the sequence of K09A-L-16-   h409A17 Humanized IgG4 version of 09A antibody comprising a heavy    chain comprising the sequence of 409A-H and a light chain comprising    the sequence of K09A-L-17-   hPD-1 human PD-1 protein-   CDR Complementarity determining region in the immunoglobulin    variable regions, defined using the Kabat numbering system-   EC50 concentration resulting in 50% efficacy or binding-   ELISA Enzyme-linked immunosorbant assay-   FW Antibody framework region: the immunoglobulin variable regions    excluding the CDR regions-   HRP Horseradish peroxidase-   IL-2 interleukin 2-   IFN interferon-   IC50 concentration resulting in 50% inhibition-   IgG Immunoglobulin G-   Kabat An immunoglobulin alignment and numbering system pioneered by    Elvin A Kabat-   mAb Monoclonal antibody-   MES 2-(N-morpholino)ethanesulfonic acid-   NHS Normal human serum-   PCR Polymerase chain reaction-   SAM sheep anti-mouse (IgG) polyclonal antibody-   V region The segment of IgG chains which is variable in sequence    between different antibodies. It extends to Kabat residue 109 in the    light chain and 113 in the heavy chain.-   VH Immunoglobulin heavy chain variable region-   VK Immunoglobulin kappa light chain variable region

“Antibody” refers to any form of antibody that exhibits the desiredbiological activity, such as inhibiting binding of a ligand to itsreceptor, or by inhibiting ligand-induced signaling of a receptor. Thus,“antibody” is used in the broadest sense and specifically covers, but isnot limited to, monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies).

“Antibody fragment” and “antibody binding fragment” mean antigen-bindingfragments and analogues of an antibody, typically including at least aportion of the antigen binding or variable regions (e.g. one or moreCDRs) of the parental antibody. An antibody fragment retains at leastsome of the binding specificity of the parental antibody. Typically, anantibody fragment retains at least 10% of the parental binding activitywhen that activity is expressed on a molar basis. Preferably, anantibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100%or more of the parental antibody's binding affinity for the target.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules, e.g., sc-Fv, unibodies (technology fromGenmab); nanobodies (technology from Domantis); domain antibodies(technology from Ablynx); and multispecific antibodies formed fromantibody fragments. Engineered antibody variants are reviewed inHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

A “Fab fragment” is comprised of one light chain and the C_(H)1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the C_(H)1and C_(H)2 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H) ² domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 and C_(H)² domains, such that an interchain disulfide bond is formed between thetwo heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

A “single-chain Fv antibody” (or “scFv antibody”) refers to antibodyfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V_(H)and V_(L) domains which enables the scFv to form the desired structurefor antigen binding. For a review of scFv, see Pluckthun (1994) THEPHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315. See also, InternationalPatent Application Publication No. WO 88/01649 and U.S. Pat. Nos.4,946,778 and 5,260,203.

A “diabody” is a small antibody fragment with two antigen-binding sites.The fragments comprises a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L) or V_(L)-V_(H)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl.Acad. Sci. USA 90: 6444-6448.

A “domain antibody fragment” is an immunologically functionalimmunoglobulin fragment containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore V_(H) regions are covalently joined with a peptide linker to createa bivalent domain antibody fragment. The two V_(H) regions of a bivalentdomain antibody fragment may target the same or different antigens.

An antibody fragment of the invention may comprise a sufficient portionof the constant region to permit dimerization (or multimerization) ofheavy chains that have reduced disulfide linkage capability, for examplewhere at least one of the hinge cysteines normally involved ininter-heavy chain disulfide linkage is altered as described herein. Inanother embodiment, an antibody fragment, for example one that comprisesthe Fc region, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC function, and/orcomplement binding (for example, where the antibody has a glycosylationprofile necessary for ADCC function or complement binding).

The term “chimeric” antibody refers to antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(See, for example, U.S. Pat. No. 4,816,567 and Morrison et al., 1984,Proc. Natl. Acad. Sci. USA 81:6851-6855).

“Humanized” forms of non-human (for example, murine) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “hypervariable region,” as used herein, refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR,” defined by sequencealignment, for example residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain; see Kabat et al., 1991,Sequences of proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. and/or thoseresidues from a “hypervariable loop” (HVL), as defined structurally, forexample, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the lightchain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy chain variable domain; see Chothia and Leskl, 1987, J. Mol. Biol.196:901-917. “Framework” or “FR” residues are those variable domainresidues other than the hypervariable region residues as herein defined.

A “human antibody” is an antibody that possesses an amino acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies disclosedherein. This definition specifically excludes a humanized antibody thatcomprises non-human antigen-binding residues.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., 1975,Nature 256:495, or may be made by recombinant DNA methods (see, forexample, U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may alsobe isolated from phage antibody libraries using the techniques describedin Clackson et al., 1991, Nature 352:624-628 and Marks et al., 1991, J.Mol. Biol. 222:581-597, for example. The monoclonal antibodies hereinspecifically include “chimeric” antibodies.

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells; natural killercells; myeloid cells, such as monocytes, macrophages, eosinophils, mastcells, basophils, and granulocytes.

As used herein, an “immunoconjugate” refers to an anti-PD-1 antibody, ora fragment thereof, conjugated to a therapeutic moiety, such as abacterial toxin, a cytotoxic drug or a radiotoxin. Toxic moieties can beconjugated to antibodies of the invention using methods available in theart.

The following nucleic acid ambiguity codes are used herein: R=A or G;Y=C or T; M=A or C; K=G or T; S=G or C; and W=A or T.

As used herein, a sequence “variant” refers to a sequence that differsfrom the disclosed sequence at one or more amino acid residues but whichretains the biological activity of the resulting molecule.

“Conservatively modified variants” or “conservative amino acidsubstitution” refers to substitutions of amino acids are known to thoseof skill in this art and may be made generally without altering thebiological activity of the resulting molecule. Those of skill in thisart recognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson, et al., Molecular Biology of theGene, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition 1987)). Suchexemplary substitutions are preferably made in accordance with those setforth below as follows:

Exemplary Conservative Amino Acid Substitutions

Original residue Conservative substitution Ala (A) Gly; Ser Arg (R) Lys,His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu(E) Asp; Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile;Val Lys (K) Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P)Ala Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V)Ile; Leu

As used herein, “% identity” between two sequences refers to a functionof the number of identical positions shared by the sequences (i.e., %homology=# of identical positions/total # of positions×100), taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm. For example, thepercent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

As used herein, the term “about” refers to a value that is within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend' in part on how the valueis measured or determined, i.e., the limitations of the measurementsystem. For example, “about” can mean within 1 or more than 1 standarddeviation per the practice in the art. Alternatively, “about” or“comprising essentially of” can mean a range of up to 20%. Furthermore,particularly with respect to biological systems or processes, the termscan mean up to an order of magnitude or up to 5-fold of a value. Whenparticular values are provided in the application and claims, unlessotherwise stated, the meaning of “about” or “comprising essentially of”should be assumed to be within an acceptable error range for thatparticular value.

“Specifically” binds, when referring to a ligand/receptor,antibody/antigen, or other binding pair, indicates a binding reactionwhich is determinative of the presence of the protein, e.g., PD-1, in aheterogeneous population of proteins and/or other biologics. Thus, underdesignated conditions, a specified ligand/antigen binds to a particularreceptor/antibody and does not bind in a significant amount to otherproteins present in the sample.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding composition, or by another cell.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of the medical condition. Effective amountalso means an amount sufficient to allow or facilitate diagnosis. Aneffective amount for a particular subject may vary depending on factorssuch as the condition being treated, the overall health of the patient,the method route and dose of administration and the severity of sideaffects. An effective amount can be the maximal dose or dosing protocolthat avoids significant side effects or toxic effects. The effect willresult in an improvement of a diagnostic measure or parameter by atleast 5%, usually by at least 10%, more usually at least 20%, mostusually at least 30%, preferably at least 40%, more preferably at least50%, most preferably at least 60%, ideally at least 70%, more ideally atleast 80%, and most ideally at least 90%, where 100% is defined as thediagnostic parameter shown by a normal subject (see, e.g., Maynard, etal. (1996) A Handbook of SOPs for Good Clinical Practice, InterpharmPress, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good ClinicalPractice, Urch Publ., London, UK).

Monoclonal Antibodies

Monoclonal antibodies to PD-1 can be made according to knowledge andskill in the art of injecting test subjects with PD-1 antigen and thenisolating hybridomas expressing antibodies having the desired sequenceor functional characteristics.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., 1990, Nature, 348:552-554. Clackson etal., 1991, Nature, 352:624-628, and Marks et al., 1991, J. Mol. Biol.222:581-597 describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., 1992, Bio/Technology, 10:779-783), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., 1993, Nuc.Acids. Res. 21:2265-2266). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

Chimeric Antibodies

The antibody DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison, et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for non-immunoglobulin material (e.g., proteindomains). Typically such non-immunoglobulin material is substituted forthe constant domains of an antibody, or is substituted for the variabledomains of one antigen-combining site of an antibody to create achimeric bivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Humanized and Human Antibodies

A humanized antibody has one or more amino acid residues from a sourcethat is non-human. The non-human amino acid residues are often referredto as “import” residues, and are typically taken from an “import”variable domain. Humanization can be performed generally following themethod of Winter and co-workers (Jones et al., 1986, Nature 321:522-525;Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988,Science 239:1534-1536), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in non-human, for example, rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework. (FR) for the humanized antibody (Sims et al., 1987, J.Immunol. 151:2296; Chothia et al., 1987, J. Mol. Biol. 196:901). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285;Presta et al., 1993, J. Immunol. 151:2623).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Humanization of antibodies is a straightforward protein engineeringtask. Nearly all murine antibodies can be humanized by CDR grafting,resulting in the retention of antigen binding. See, Lo, Benny, K. C.,editor, in Antibody Engineering: Methods and Protocols, volume 248,Humana Press, New Jersey, 2004.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovitset al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year inImmunology 7:33; and Duchosal et al., 1992, Nature 355:258. Humanantibodies can also be derived from phage-display libraries (Hoogenboomet al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991,222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., 1992, Bio/Technology 10:163-167 describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc region that is present in the antibody. Protein A canbe used to purify antibodies that are based on human .gamma.1, .gamma.2,or .gamma.4 heavy chains (Lindmark et al., 1983, J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and forhuman.gamma.3 (Guss et al., 1986, EMBO J 5:15671575). The matrix towhich the affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In one embodiment, the glycoprotein may be purified using adsorptiononto a lectin substrate (e.g. a lectin affinity column) to removefucose-containing glycoprotein from the preparation and thereby enrichfor fucose-free glycoprotein.

Pharmaceutical Formulations

The invention comprises pharmaceutical formulations of a PD-1 antibodyor antibody fragment of the invention. To prepare pharmaceutical orsterile compositions, the antibody or fragment thereof is admixed with apharmaceutically acceptable carrier or excipient, see, e.g., Remington'sPharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, MackPublishing Company, Easton, Pa. (1984). Formulations of therapeutic anddiagnostic agents may be prepared by mixing with physiologicallyacceptable carriers, excipients, or stabilizers in the form of, e.g.,lyophilized powders, slurries, aqueous solutions or suspensions (see,e.g., Hardman, et al. (2001) Goodman and Gilman's The PharmacologicalBasis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000)Remington: The Science and Practice of Pharmacy, Lippincott, Williams,and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker,NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000)Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with an immunosuppressive agent,can be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in humans. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.

Suitable routes of administration include parenteral administration,such as intramuscular, intravenous, or subcutaneous administration andoral administration. Administration of antibody used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral, intraarterial or intravenousinjection. In one embodiment, the binding compound of the invention isadministered intravenously. In another embodiment, the binding compoundof the invention is administered subcutaneously.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto the site of action, often in a depot or sustained releaseformulation. Furthermore, one may administer the antibody in a targeteddrug delivery system.

Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available (see, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003)New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh, etal. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000) New Engl.J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, oneweek, or 1-7 times per week. Doses may be provided intravenously,subcutaneously, intraperitoneally, cutaneously, topically, orally,nasally, rectally, intramuscular, intracerebrally, intraspinally, or byinhalation. A preferred dose protocol is one involving the maximal doseor dose frequency that avoids significant undesirable side effects. Atotal weekly dose is generally at least 0.05 μg/kg body weight, moregenerally at least 0.2 μg/kg, most generally at least 0.5 μg/kg,typically at least 1 μg/kg, more typically at least 10 μg/kg, mosttypically at least 100 μg/kg, preferably at least 0.2 mg/kg, morepreferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg,optimally at least 10 mg/kg, more optimally at least 25 mg/kg, and mostoptimally at least 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J.Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji,et al. (20003) Cancer Immunol. Immunother. 52:133-144). The desired doseof a small molecule therapeutic, e.g., a peptide mimetic, naturalproduct, or organic chemical, is about the same as for an antibody orpolypeptide, on a moles/kg basis.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with diseaseand/or a reduction in the severity of such symptoms that will or areexpected to develop with said disease. The terms further includeameliorating existing symptoms, preventing additional symptoms, andameliorating or preventing the underlying causes of such symptoms. Thus,the terms denote that a beneficial result has been conferred on avertebrate subject with a disease.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of an anti-PD-1 antibody orfragment thereof, that when administered alone or in combination with anadditional therapeutic agent to a cell, tissue, or subject is effectiveto prevent or ameliorate the disease or condition to be treated. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously. An effective amount oftherapeutic will decrease the symptoms typically by at least 10%;usually by at least 20%; preferably at least about 30%; more preferablyat least 40%, and most preferably by at least 50%.

Methods for co-administration or treatment with a second therapeuticagent are well known in the art, see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., PA.

The pharmaceutical composition of the invention may also contain otheragent, including but not limited to a cytotoxic, cytostatic,anti-angiogenic or antimetabolite agent, a tumor targeted agent, animmune stimulating or immune modulating agent or an antibody conjugatedto a cytotoxic, cytostatic, or otherwise toxic agent. The pharmaceuticalcomposition can also be employed with other therapeutic modalities suchas surgery, chemotherapy and radiation.

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

Therapeutic Uses for the Antibody and Antibody Fragments of theInvention

The antibody or antigen binding fragments of the invention, whichspecifically bind to human PD-1, can be used to increase, enhance,stimulate or up-regulate an immune response. The antibodies and antibodyfragments of the invention are particularly suitable for treatingsubjects having a disorder that can be treated by increasing the T-cellmediated immune response. Preferred subjects include human patients inneed of enhancement of an immune response.

Cancer

The antibody or antigen binding fragments of the invention can be usedto treat cancer (i.e., to inhibit the growth or survival of tumorcells). Preferred cancers whose growth may be inhibited using theantibodies of the invention include cancers typically responsive toimmunotherapy, but also cancers that have not hitherto been associatedwith immunotherapy. Non-limiting examples of preferred cancers fortreatment include melanoma (e.g., metastatic malignant melanoma), renalcancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormonerefractory prostate adenocarcinoma), pancreatic adenocarcinoma, breastcancer, colon cancer, lung cancer (e.g. non-small cell lung cancer),esophageal cancer, squamous cell carcinoma of the head and neck, livercancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma,glioma, leukemia, lymphoma, and other neoplastic malignancies.Additionally, the invention includes refractory or recurrentmalignancies whose growth may be inhibited using the antibodies of theinvention.

The antibody or antibody fragments of the invention can be used alone orin combination with: other anti-neoplastic agents or immunogenic agents(for example, attenuated cancerous cells, tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), antigenpresenting cells such as dendritic cells pulsed with tumor derivedantigen or nucleic acids, immune stimulating cytokines (for example,IL-2, IFNa2, GM-CSF_), and cells transfected with genes encoding immunestimulating cytokines such as but not limited to GM-CSF); standardcancer treatments (for example, chemotherapy, radiotherapy or surgery);or other antibodies (including but not limited to antibodies to VEGF,EGFR, Her2/neu, VEGF receptors, other growth factor receptors, CD20,CD40, CTLA-4, OX-40, 4-IBB, and ICOS).

Infectious Diseases

The antibody or antibody fragments of the invention can also be used toprevent or treat infections and infectious disease. The antibody orantibody fragments can be used alone, or in combination with vaccines,to stimulate the immune response to pathogens, toxins, andself-antigens. The antibodies or antigen-binding fragment thereof can beused to stimulate immune response to viruses infectious to humans, suchas, but not limited to, human immunodeficiency viruses, hepatitisviruses class A, B and C, Eppstein Barr virus, human cytomegalovirus,human papilloma viruses, herpes viruses. The antibodies orantigen-binding fragment thereof can be used to stimulate immuneresponse to infection with bacterial or fungal parasites, and otherpathogens.

Vaccination Adjuvant

The antibody or antibody fragments of the invention can be used inconjunction with other recombinant proteins and/or peptides (such astumor antigens or cancer cells) in order to increase an immune responseto these proteins (i.e., in a vaccination protocol).

For example, anti-PD-1 antibodies and antibody fragments thereof may beused to stimulate antigen-specific immune responses by coadministrationof an anti-PD-1 antibody with an antigen of interest (e.g., a vaccine).Accordingly, in another aspect the invention provides a method ofenhancing an immune response to an antigen in a subject, comprisingadministering to the subject: (i) the antigen; and (ii) an anti-PD-1antibody of the invention or antigen-binding portion thereof, such thatan immune response to the antigen in the subject is enhanced. Theantigen can be, for example, a tumor antigen, a viral antigen, abacterial antigen or an antigen from a pathogen. Non-limiting examplesof such antigens include, without limitation, tumor antigens, orantigens from the viruses, bacteria or other pathogens.

Th2 Mediated Diseases

Anti-PD-1 antibodies and antibody fragments of the invention can also beused to treat Th2 mediated diseases, such as asthma and allergy. This isbased on the finding that the antibodies of the invention can helpinduce a Th1 response. Thus, the antibodies of the invention can be usedto in Th2 mediated diseases to generate a more balanced immune response.

Ex-Vivo Activation of T Cells

The antibodies and antigen fragments of the invention can also be usedfor the ex vivo activation and expansion of antigen specific T cells andadoptive transfer of these cells into recipients in order to increaseantigen-specific T cells against tumor. These methods may also be usedto activate T cell responses to infectious agents such as CMV. Ex vivoactivation in the presence of anti-PD-1 antibodies may be expected toincrease the frequency and activity of the adoptively transferred Tcells.

Other Combination Therapies

As previously described, anti-PD-1 antibodies of the invention can becoadministered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunocomplex) or can beadministered separately from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies.

Antibodies and antigen binding fragments of the invention can also beused to increase the effectiveness of donor engrafted tumor specific Tcells.

Non-Therapeutic Uses for the Antibody and Antibody Fragments of theInvention

A market for anti-PD-1 antibodies for non-therapeutic uses alreadyexists, as demonstrated by the commercial sales of J116, and J105monoclonal anti-hPD-1 antibodies sold by eBioscience of San Diego,Calif., USA, for use in flow cytometric analysis, immunohistochemistryand in vitro functional assays; and mab1086, a monoclonal anti-hPD-1antibody sold by R&D Systems of Minneapolis, Minn., USA, for use in flowcytometry, Western blots and ELISA. Antibodies of the invention may beused for any non-therapeutic purpose now served by J116, J105 and/orMab1086.

The antibody of the invention may be used as an affinity purificationagent.

The antibody may also be useful in diagnostic assays, e.g., fordetecting expression of PD-1 in specific cells, tissues, or serum. Fordiagnostic applications, the antibody typically will be labeled (eitherdirectly or indirectly) with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:biotin, fluorochromes, radionucleotides, enzymes, iodine, andbiosynthetic labels.

The antibody of the present invention may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies. AManual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibody may also be used for in vivo diagnostic assays. Generally,the antibody is labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ⁴C,³1I, ¹²⁵I, ³H, ³²P ³⁵S or ¹⁸F) so that the antigen or cells expressingit can be localized using immunoscintiography or positron emissiontomography.

Deposit of Materials

DNA constructs encoding the variable regions of the heavy and lightchains of the humanized antibodies h409A11, h409A16 and h409A17 havebeen deposited with the American Type Culture Collection PatentDepository (10801 University Blvd., Manassas, Va.). The plasmidcontaining the DNA encoding the heavy chain of h409A-11, h409A-16 andh409A-17 was deposited on Jun. 9, 2008 and identified as 081469_SPD-H.The plasmid containing the DNA encoding the light chain of h409A11 wasdeposited on Jun. 9, 2008 and identified as 0801470_SPD-L-11. Theplasmid containing the DNA encoding the light chain of h409A16 wasdeposited on Jun. 9, 2008 and identified as 0801471_SPD-L-16. Theplasmid containing the DNA encoding the light chain of h409A17 wasdeposited on Jun. 9, 2008 and was designated 0801472_SPD-L-17. Thedeposits were made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty).

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the culture deposited, sincethe deposited embodiment is intended as a single illustration of oneaspect of the invention and any culture that is functionally equivalentis within the scope of this invention. The deposit of material hereindoes not constitute an admission that the written description hereincontained is inadequate to enable the practice of any aspect of theinvention, including the best mode thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustration that itrepresents. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of this invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

EXAMPLES Example 1 Immunization and Selection of Anti PD-1 Antibodies

Immunization of Mice with hPD-1 cDNA

To generate antibodies against the human PD-1 (‘hPD-1’) receptor, a cDNAencoding the open reading frame of the hPD-1 receptor was obtained byPCR and subcloned into vector pcDNA3.1 (Invitrogen, Carlsbad, Calif.).Next, CHO-K1 cells were stably transfected with hPD-1, and expressionwas monitored using flow cytometry. CHO-K1 clones were isolatedexpressing human PD-1 on their membranes and named CHO-hPD1.

Mice were immunized by gene gun immunization using a Helios gene gun(BioRad) and DNA coated gold bullets (BioRad) following manufacturersinstructions. Briefly, 1 μm gold particles were coated with hPD-1 cDNA(cloned into pcDNA3.1) and, where indicated, commercial expressionvectors for mouse Flt3L and mouse GM-CSF in a 2:1:1 ratio (both fromAldevron, Fargo N. Dak.). A total of 1 μg of plasmid DNA was used tocoat 500 μg of gold bullets.

Specifically, 7-8 week-old female BALB/C mice were immunized on the earby gene gun receiving 2, 3, or 4 cycles of a shot on both ears (seeTable I). One mouse received a final booster with 5×10⁶ CHO-hPD1 cellsin the peritoneal cavity. Approximately, a 1:1000 anti-hPD-1 titers wasdetectable in mouse serum after two DNA immunizations by cell ELISAusing CHO-hPD-1 versus CHO-K1 parental cells. Four days after the finalimmunization, mice were sacrificed, and erythrocyte-depleted spleen cellpopulations were prepared as described previously (Steenbakkers et al.,1992, J. Immunol. Meth. 152:69-77; Steenbakkers et al., 1994, Mol. Biol.Rep. 19:125-134) and frozen at −140° C.

TABLE I Immunization schedule used to induce hPD-1 specific antibodytiters in Balb/c mice. Week 1 Week 4 Week 7 Week 8 Week 9 Week 10 Week11 Mouse 730 2 shots hPD1 2 shots 2 shots Harvest of pCDNA3.1 hPD1 hPD1spleen cells pCDNA3.1 pCDNA3.1 Mouse 731 2 shots hPD1 4 shots 5 millionHarvest of pCDNA3.1 hPD1 CHO-hPD1 spleen cells pCDNA3.1 Mouse738 2 shotshPD1 2 shots 2 shots 2 shots Harvest pCDNA3.1 hPD1 hPD1 hPD1 of spleen(mFlt3L + pCDNA3.1 pCDNA3.1 pCDNA3.1 cells mGM-CSF) (mFlt3L + (mFlt3L +(mFlt3L + mGM-CSF) mGM-CSF) mGM-CSF)

Selection of Anti-PD-1 Antibody Producing B Cells

To select B cell clones producing anti-human-PD-1 antibodies, 2×10⁷erythrocyte-depleted spleen cells from hPD-1 DNA immunized mice, i.e.,mouse 730, 731 and 738 (see Table I), were pooled for a B-cell culture.Spleen cells were incubated in DMEM/HAM's F12/10% Calf Serum (Hyclone,Logan, Utah, USA) for one hour at 37° C. in a plastic culture flask toremove monocytes. Non-adherent cells were submitted to one round ofnegative panning on CHO-K1 cells, followed by positive panning onCHO-hPD1 cells. Both selection procedures were performed for one hour at37° C. on confluently grown cultures in 21 cm² petridishes or T25culture flasks (cell cultures were irradiated before use, to a totaldose of 2000 RAD). After the positive panning, unbound cells wereremoved by washing ten times with PBS supplemented with 0.132%CaCl₂.2H₂0 and 0.1% MgCl₂.6H₂0. Finally, bound B-cells were harvested bytrypsin treatment.

Selected B-cells were cultured and immortalized as described inSteenbakkers et al., 1994, Mol. Biol. Rep. 19:125-134. Briefly, selectedB cells were mixed with 7.5% (v/v) T-cell supernatant and 50,000irradiated (2,500 RAD) EL-4 B5 nursing cells in a final volume of 200 μLDMEM/HAM's F12/10% Bovine Calf Serum, in 96-well flat-bottomed tissueculture plates. On day eight, supernatants were screened for theiranti-hPD-1 reactivity by CHO-hPD-1 cell ELISA using the followingprocedure. CHO-K1 and CHO-hPD1 cells were cultured to confluency inflatbottom 96-well plates in 50 μL DMEM/HAM'S F12, 10% FBS. Next, 50 μLof immunoglobulin-containing supernatant was added for 1 hr at 37° C.After three washes with PBS-Tween, 100 μL (1:1000 diluted)goat-anti-mouse-horseradish peroxidase (HRP, Southern, Birmingham, Ala.,USA) in DMEM/HAM'S F12/10% FBS was added for 1 hour at 37° C. Afterthree washes with PBS-Tween, immobilized immunoglobulins were visualizedwith UPO/TMB (Biomerieux, Boxtel, Netherlands).

From this B-cell culture, 13 hPD-1 reactive supernatants were identifiedand shown to inhibit Jurkat T cell activation when immobilized onplastic, and B-cell clones from positive wells were immortalized bymini-electrofusion following published procedures (Steenbakkers et al.,1992, J. Immunol. Meth. 152:69-77; Steenbakkers et al., 1994, Mol. Biol.Rep. 19:125-134). Specifically, B-cells were mixed with 10⁶ NS-1 myelomacells, and serum was removed by washing with DMEM/HAM's F12. Next, cellswere treated with pronase solution for three minutes and subsequentlywashed with fusion medium. Electrofusion was performed in a 50 μL fusionchamber by an alternating electric field of 30 s, 2 MHz, 400 V/cmfollowed by a square, high field pulse of 10 μs, 3 kV/cm and again analternating electric field of 30 s, 2 MHz, 400 V/cm. Finally, thecontent of the fusion chamber was transferred to hybridoma selectionmedium and plated into a 96-well plate under limiting dilutionconditions. On day 14 after fusion, the cultures were examined forhybridoma growth and screened for the presence of antibody reactivity tohPD-1. This procedure yielded five different anti-hPD-1 hybridomas,named hPD-1.05A, hPD-1.06B, hPD-1.08A, hPD-1.09A and hPD-1.13A, thatwere subcloned by limiting dilution to safeguard their integrity andfurther cultured to produce antibody. Supernatants obtained from thesehybridomas strongly inhibited the IL-2 production from Jurkat E6.2.11cells upon anti-CD3/anti-CD28 stimulation (see FIG. 1 and text below).

Jurkat E6.1 cells (American Type Culture Collection) were subcloned bylimiting dilution using standard methodology and subclones were testedfor enhanced capacity to produce IL-2 upon cross-linking of CD3 andCD28. A high IL-2 producing subclone was obtained and subsequently namedJurkat E6.2.11 and used in further assays. Costar 3370 96-well assayplates were coated overnight at 4° C. with 5 μg/mL Sheep Anti-Mouse Ig(SAM). Excess of SAM was removed and plates were blocked for 1 hr atroom temperature with 200 μL/well PBS/10% Fetal Bovine Serum. Afterthree washes with PBS, wells were coated with 100 μL/well anti-CD3(OKT3; 10 or 60 ng/mL) for 1 hr at 37° C. After three washes with PBS,50 μL/well PBS/10% Fetal Bovine Serum and 50 μL/well B-cell- orhybridoma supernatant was added for 30 min at 37° C. After three washeswith PBS, 120 μL/well of cell suspension, Jurkat E6.2.11 cells (2×10⁵cells/well+0.5 μg/mL anti-CD28 (Sanquin #M1650, Central Laboratory forBloodtransfusion, Amsterdam, NL) in DMEM/F12/10% Fetal Bovine Serum) wasadded. After a 6 h culture, supernatant was examined for IL-2 productionusing a standard sandwich ELISA with anti-hIL-2 capture and biotinylateddetection antibody pairs from Pharmingen and Streptavidin-Horse RadishPeroxidase (Southern Biotech) as a detection reagent. To determine thepotency of these antibodies as compared with PD-L1, a small group ofmAbs was produced on a larger scale. The mAbs were purified usingProtein G affinity chromatography (see Example 2). Purified antibodies,hPD-L1/Fc (recombinant human B7-H1/Fc chimera, R&D systems) or mouseIgG1 kappa (from Sigma) as a negative control were coated at identicalconcentrations on plates with anti-CD3 as described above. JurkatE6.2.11 cells and anti-CD28 were added for six hours, and T-cellactivation was measured by IL-2 produced in the supernatant. Two of theantibodies (hPD1.08A and hPD1.09A) showed an 8-10 fold more potentinhibition compared to immobilized PD-L1/Fc.

Example 2 Purification and Characterization of Murine Anti-PD-1Antibodies Stabilization of Anti-PD-1 Producing Hybridomas andPurification of Anti-PD-1 Antibodies

Clonal cell populations were obtained for each of the hybridomas bysubjecting them to multiple rounds (>4) of limiting dilution. Stablehybridoma cells were then cultured under serum-free conditions usingCELLine bioreactors (Integra-biosciences) for six to eight days. Cellswere seeded in the inner chamber in serum-free media at a density of3×10⁶ c/mL in 15 mL and expanded to approximately 4×10⁷ c/mL over eightdays. The outer chamber was filled with media supplemented with up to10% BCS (bovine calf serum). On day six to eight, the inner chamberculture was harvested, washed with 15 mL SF media and re-innoculatedwith hybridoma cells. Bioreactor supernatant and wash were combined andclarified by centrifugation. The resulting supernatant was filteredthrough a 0.22 μM filter membrane. For antibody purification,supernatants were diluted 1:1 in high salt binding buffer (1M Glycine/2MNaCl, pH 9.0), and mAbs were purified using Protein G HiTrap 5 mL column(GE healthcare). After washing with PBS, bound antibodies were elutedusing 0.1 M Glycine pH=2.7, followed by pH neutralization using 3 MTris. Finally, the buffer was exchanged for PBS using PD-10gel-filtration columns (GE healthcare), and antibodies were concentratedusing Ultra-15 centrifugal concentrators (Amicon) and quantified usingspectrophotometry.

Commercial Antibodies

The following commercial antibodies were used in various studiesdescribed herein: Anti-PD-1 antibody clone J116 (#14-9989) was purchasedfrom eBioscience. Anti-CTLA-4 clone 14D3 (mAb 16-1529) was purchasedfrom eBioscience. Anti-PD-1 clone 192106 (mAb1086) was purchased fromR&D systems (#mAb1086). Isotype control antibody mIgG1, kappa, cloneMOPC21 was purchased from Sigma (#M9269). Isotype controls mIgG1 kappa(mAb 16-4714) and IgG2a kappa (mAb 16-4724) were purchased fromeBioscience.

Binding Analysis

Protein-based and cell-based ELISA (‘CELISA’) experiments were used todetermine apparent binding affinities (reported as EC50 values). In somecases, the binding of the anti-PD-1 antibodies was compared to that ofcommercial anti-PD-1 antibodies J116 (eBiosciences) and Mab1086 (R&Dsystems).

A protein ELISA was used for determination of the relative binding ofantibodies to human PD-1/Fc. hPD-1/Fc (R & D Systems) was immobilizedonto Maxisorp 96-well plates (Nunc) by incubation for 4 h at roomtemperature (or overnight at 4° C.). Nonspecific binding sites wereblocked by incubation with 3% BSA in PBST for one hour at roomtemperature. After coating, the plates were washed three times withPBST. Dilutions of anti-PD-1 antibodies were prepared in binding buffer(PBS containing 0.1% Tween 20 and 0.3% BSA) and incubated with theimmobilized fusion protein for one hour at 25° C. After binding, theplates were washed three times with PBST, incubated for one hour at 25°C. with peroxidase-labeled goat anti-mouse IgG (Southern Biotech)diluted 1/4,000 in binding buffer, washed again, and developed usingTMB. ELISA results are shown in FIG. 2. The concentration ofhalf-maximal binding is reported as a measure of relative bindingaffinity (Table II).

Binding to CHO-hPD-1 cells was also assessed by CELISA. For CELISA,CHO-hPD-1 cells were cultured to 80 to 100 percent confluency in 50 μLculture medium (DMEM/HAM'S F12, 10% FBS). Next, 50 μL media containingvarious concentrations of purified mAb were added for one hour at 37° C.After three washes with PBS-Tween, 100 μL goat-anti-mouse-HRP (SouthernBiotech cat #1030-05) (diluted 1:1000 in culture medium) was added forone hour at 37° C. After three additional washes with PBS-Tween,immobilized immunoglobulins were visualized with colorimetric peroxidasesubstrate TMB (BD Biosciences). Absorbance increase due to peroxidaseactivity (450 nm) was measured in a microtiter plate reader. FIG. 2shows the dose-response relation between concentration and binding forantibodies hPD-1.08A and hPD-1.09A. The results of the protein and cellbinding studies are summarized in Table II.

Kinetic Analysis by Bio-Light Interferometry (ForteBio)

To further characterize the binding characteristics of the antibodies,each was profiled using bio-light interferometry on the Octet system(ForteBio, Menlo Park, Calif.) to elucidate binding kinetics andcalculate equilibrium binding constants. This assay was, performed bycoupling PD-1-Fc fusion protein (R&D Systems) to amine-reactivebiosensors (Fortebio) using standard amine chemistry. Anti-PD-1 mAbbinding to and dissociation from the biosensors was then observed atvarious antibody concentrations. Specifically, amine-reactive biosensorswere pre-wet by immersing them in wells containing 0.1M MES pH=5.5 for 5minutes. The biosensors were then activated using a 0.1M NHS/0.4M EDCmixture for 5 minutes. PD-1/Fc fusion protein (R & D systems) wascoupled by immersing the biosensors in a solution of 12 ug/mL PD-1/Fc in0.1M MES for 7.5 minutes. The biosensor surface was quenched using asolution of 1M ethanolamine for 5 minutes. Biosensors were equilibratedin PBS for 5 minutes. Association of anti-PD-1 mAbs was observed byplacing the biosensors in wells containing various antibodyconcentrations (10-80 nM purified antibody >99% by SDS-PAGE in PBS) andmonitoring interferometry for 30 minutes. Dissociation was measuredafter transfer of the biosensors into PBS and monitoring of theinterferometry signal for 60 minutes. The observed on and off rates(k_(obs) and k_(d)) were fit using a 1:1 binding global fit modelcomprising all concentrations tested, and the equilibrium bindingconstant K_(D) was calculated. Results from the kinetic studies arepresented in Table II, and FIG. 6 below.

TABLE II Biochemical characterization summary of murine anti-PD-1 mAbs.Binding Analysis Ligand Blockade Kinetic Analysis ELISA CELISA FACS FMATFortebio Octet EC50 (pM) EC50 (pM) IC50 (pM) IC50 (pM) k_(assoc)k_(dissoc) K_(D) mAb hPD-1/Fc hPD-1/CHO PD-L1 PD-L1 PD-L2 1/s 1/Ms M 05A338 15 1.62E+05 1.11E−04 6.90E−10 06B 135 160 8.32E+04 9.74E−05 1.17E−0908A 76 79 0.9 0.73 2.1 1.25E+06 3.03E−05 2.41E−11 09A 123 113 0.8 0.901.7 1.64E+06 3.60E−05 2.20E−11 13A 485 64 1.46E+05 4.16E−04 2.85E−09J116 410 349 106 >100 44 8.24E+04 1.50E−04 1.82E−09 mAb108659 >10000 >10000 >10000 >10000 2.45E+05 1.68E−04 6.86E−10

Two of the monoclonal antibodies, hPD-1.08A and hPD-1.09A, boundconsiderably more tightly than any other mAb tested using this assay,with K_(D) determined to be 24 and 22 pM for hPD-1.08A and hPD-1.09A,respectively. Compared to the other anti-PD-1 antibodies tested, theincreased affinity is due to a slower off-rate and a significantlyfaster on-rate measured for hPD-1.08A and hPD-1.09A.

Ligand Blockade

Blockade of ligand binding studied using flow cytometry. CHO cellsexpressing human PD-1 were dissociated from adherent culture flasks andmixed with varying concentrations of anti-PD-1 antibody and a constantconcentration (600 ng/mL) of unlabeled hPD-L1/Fc or recombinant humanPD-L2/Fc fusion protein (both from R&D Systems) in a 96-well plate. Themixture was equilibrated for 30 minutes on ice, washed three times withFACS buffer (PBS containing 1% BCS and 0.1% sodium azide), and incubatedwith FITC labeled goat anti-human Fc for a further 15 minutes on ice.The cells were washed again with FACS buffer and analyzed by flowcytometry. Data were analyzed with Prism (GraphPad Software, San Diego,Calif.) using non-linear regression, and 1050 values were calculated.

Calculated IC₅₀ data are summarized in Table II. Antibodies 05A, 06B and13A were determined to demonstrate a K_(D) between 600 μM and 3 nM forthe binding of hPD-1. Despite the tight binding, these antibodies eachdemonstrated IC₅₀>10 nM for the blockade of hPD-L1 binding to hPD-1. Thecommercially available anti-PD-1 antibody J116 (eBiosciences) weaklycompeted with PD-L1 for binding, having a calculated IC50 outside therange of this experiment (>100, nM). Control mouse IgG1 does not competewith PD-L1 for PD-1 binding. In contrast, the high affinity antibodieshPD-1.08A and hPD-1.09A inhibited PD-L1 binding with IC₅₀ values below 1nM, whereas PD-L2 binding was blocked with IC₅₀ values around 1-2 nM(Table II). PD-L2 was reported earlier to bind to PD-1 with a two- tosix-fold higher affinity than does PD-L1 (Youngnak P. et al., 2003,Biochem. Biophys. Res. Commun. 307, 672-677).

Ligand blockade was confirmed using a homogeneous competition assay anddetection using fluorometric microvolume assay technology (FMAT).Briefly, CHO.hPD-1 were dissociated from adherent culture flasks, mixedwith varying concentrations of anti-PD-1 antibody and a constantconcentration (600 ng/mL) of hPD-L1/Fc or hPD-L2/Fc fusion protein (bothfrom R&D Systems), labeled with a fluorescent dye (AlexaFluor 647,Invitrogen) in a 96-well plate. The mixture was equilibrated for 90minutes at 37° C. and read using an AB8200 Cellular Detection Analyzer(Applied Biosystems, Foster City, Calif.). Data was analyzed with Prism(GraphPad Software, San Diego, Calif.) using non-linear regression, andIC₅₀ values were calculated. FIG. 3 shows results of a dose-responseexperiment indicating that the magnitude of ligand blockade isdetermined by antibody concentration. Binding of both hPD-L1/Fc andhPD-L2/Fc to CHO-hPD-1 cells can be completely inhibited by hPD-1.08A,hPD-1.09A and (to a lesser extent) by J116 in a dose-dependent fashion.Calculated IC₅₀ data are summarized in Table II. Confirming the resultsobtained using flow cytometry, the high affinity antibodies hPD-1.08Aand hPD-1.09A inhibited PD-L1 binding with IC₅₀ values below 1 nM.

Species Cross-Reactivity

To assess the species cross-reactivity of the antibodies, the mouse andcynomolgus macaque PD-1 receptors were cloned by PCR and stablytransfected CHO-K1 cells were generated. The antibodies were tested forbinding to the cynomolgus receptor using a CELISA. Commercial antibodyJ116, hPD-1.08A and hPD-1.09A were found to bind with equal affinity tohuman and cynomolgus PD-1 and block binding of hPD-L1/Fc and hPD-L2/Fcto cynomolgous PD-1 with similar efficacy as compared to human PD-1.This is not surprising because the amino acid sequence of theextracellular portion of cynomolgus PD-1 was found to be 97% identicalto that of human PD-1. In addition to PD-1 from cynomolgous macaques,hPD-1.08A and hPD-1.09A also functionally blocked PD-1 from rhesusmacaques in SEB stimulated blood cell cultures described in Example 3.None of the antibodies tested bound mouse PD-1 with detectable affinityin any of the assays used.

In summary, five anti-PD-1 monoclonal antibodies were purified andcharacterized, which were isolated based on their ability to modulateJurkat function. These antibodies bound tightly to PD-1 (withdissociation constants in the 20 μM to 3 nM range) and were capable ofblocking the interaction with both PD-L1 and PD-L2 with varying IC50values. Four of these anti-hPD-1 mAbs were considerably better than thebest available commercial anti-PD-1 mAbs. Each of the antibodies, whenadded in solution acted as receptor antagonists, ultimately enhancing Tcell responses (see Example 3).

Example 3 Functional Profiling of Anti-PD-1 Antibodies

Human T Cell Response to SEB is Enhanced by hPD-1.08A and hPD-1.09A

Anti-PD-1 antibodies were tested for their capacity to enhance T cellactivity in vitro using blood cells from healthy volunteers. One assayused to characterize the functional consequence of blocking human PD-1receptor utilized Staphylococcus enterotoxin B (SEB) to engage andactivate all T cells expressing the Vβ3 and Vβ8 T cell receptor chain.Healthy human donor blood was obtained and diluted 1:10 into culturemedium. Diluted whole blood was plated (150 μl per well) in 96-wellround-bottom plates and pre-incubated for 30-60 min with mAb and varyingconcentrations. SEB was then added at various concentrations rangingfrom 10 ng/mL to 10 μg/mL. Supernatants were collected after 2 to 4 daysof culture and the amount of IL-2 produced was quantified using ELISA(described in Example 1) or using standard multiplex technology (Luminexplatform—Biosource cytokine detection kits). Titration of SEB from 100ng/mL up to 10 μg/mL significantly stimulated IL-2 production bywhole-blood cells. Usually, depending on the donor, 100 to 1000 pg/mLIL-2 was detectable by ELISA 2-4 days after stimulation with 1 μg/mL ofSEB. Addition of hPD-1.08A and hPD-1.09A enhanced IL-2 production overcontrol mouse IgG1, on average 2 to 4 fold at the highest antibodyconcentration tested (25 μg/mL). The stimulation index was averaged forexperiments performed with a set of independent healthy volunteers (FIG.4). These experiments demonstrated that both hPD-1.08A and hPD-1.09Aenhanced IL-2 production upon SEB stimulation of diluted whole-bloodcells. Both PD-1 and PD-L1 (but not PD-L2) expression levels wereupregulated (quantified by flow cytometry) over time after SEBstimulation of whole blood cells. Anti-PD-L1 monoclonal antibody (cloneMIH5, Ebiosciences #16-5982) and anti-CTLA-4 (clone 14D3, eBiosciences#16-1529) also induced an increase in IL-2 production under similarconditions, a finding that further validated the use of the SEBstimulation assay to quantify T cell activity after manipulation ofcostimulatory pathways (FIG. 4). The enhanced IL-2 production byanti-PD-1 antibodies was found to be dose-dependent. In addition toIL-2, by Luminex technology levels of TNFα, IL-17, IL-7, IL-6 and IFNγwere also found to be significantly modulated by hPD-1.08A andhPD-1.09A. The results of these experiments indicate that hPD-1.08A andhPD-1.09 can be used to stimulate human T cell responses.

Anti-PD-1 antibody, hPD-1.09A, was further tested for its capacity toenhance T cell activity in vitro using blood cells derived from cancerpatients. Blood from patients with advanced melanoma (1 patient) orprostate cancer (3 patients) was tested following the above protocol.Results of the cytokine quantitation are presented in Table III as foldincrease of cytokine produced when cells are stimulated in the presenceof 25 ug/mL hPD-1.09A compared to SEB stimulation in the absence ofantibody. In summary, hPD-1.09A was found to increase the SEB inducedIL-2 production 2 to 3.5 fold for each of the 4 patients. Similarlyproduction of TNFα, IL-17 and IFNγ was enhanced, and production of IL-5and IL-13 was decreased. These experiments indicate that hPD-1.09A hasthe ability to stimulate T cell responses in cancer patients. Further,these experiments suggest a preference towards Th1 responses.

TABLE III SEB-stimulated cytokine production in the presence ofhPD-1.09A cancer Fold change in cytokine level patient type IL-2 TNFαIFNγ IL-5 IL-6 IL-13 IL-17 A prostate 3.4 2.0 1.9 0.7 2.1 0.8 1.8 Bprostate 2.1 1.5 1.2 0.4 2.2 0.6 2.6 C prostate 2.0 2.4 2 0.9 2.4 1.12.4 D melanoma 2.0 1.9 1.5 0.4 1.9 0.5 2.0Human Recall T Cell Response to TT Challenge is Enhanced by hPD-1.08Aand hPD-1.09A

Another assay used to profile the functional effect of anti-human PD-1antibodies blocking receptor interaction with its natural ligands usedthe tetanus toxoid (TT) antigen to stimulate pre-existing memory T cellsin healthy donor blood. To this end, freshly prepared PBMC (2×10⁵ cells)were plated in 96 well round-bottom plates in complete RPMI 1640 medium(containing 5% heat inactivated human serum), pre-incubated with testantibodies at varying concentration and stimulated with TT (AstarteBiologics) at a concentration of 100 ng/mL. The cells were incubated for3-7 days at 37° C., 5% CO₂ after which supernatants were harvested.Cytokine concentrations were determined by ELISA (IL-2 and IFN-γ ELISAdetection antibody pair sets from eBioscience) and multiplex analysis(Luminex platform—Biosource cytokine detection kits). Blockade of PD-1enhanced proliferation and significantly enhanced cytokine production(FIG. 5) including IFNγ and IL-2 compared to antigen alone. Luminexanalysis revealed that production of the cytokines GM-CSF, RANTES, andIL-6 are increased upon PD-1 blockage.

Staining of Human PD-1 on Formalin-Fixed Paraffin-Embedded Human Cells

Since SEB-stimulated blood cells demonstrated enhanced expression ofPD-1 by flow cytometry, these cells were used to determine if hPD-1.09Acould detect PD-1 in formalin-fixed paraffin embedded tissue forhistological use. Human donor peripheral blood mononuclear cells werestimulated with 0.1 μg/mL SEB for 3 days, after which the non-adherentcells (mainly lymphocytes) were collected, washed twice with PBS andcentrifuged (1100 rpm for 5 min.). The cells were fixed for 10 min in 4%formaldehyde, the cell-pellet was embedded in agarose, dehydrated inethanol (subsequently 70%, 80%, 96% and 100%) and xylene, and thereafterembedded in paraffin. Sections (4 μm) were mounted onto glass slides andhydrated (xylene, ethanol 100%, 96%, 80%, 70%, PBS buffer), after whichantigen retrieval in heated citrate buffer was performed using standardmethodology. Peroxidase activity was blocked using 100% methanolincluding 0.3% H₂O₂ and slides were rinsed in water and PBS, Tween 0.1%.Sections were incubated with hPD-1.09A for 1.5 hours at roomtemperature, rinsed with PBS-Tween, followed by standard detectionmethods. Slides were counterstained with hematoxylin for 30 seconds atroom temperature, dehydrated with xylene, and mounted for microscopicalexamination. These experiments showed that lymphocytes derived from SEBstimulated PBMC cultures stained strongly (when compared to the isotypecontrol) with hPD-1.09A, as opposed to unstimulated PBMC cultures,indicating that hPD-1.09A is useful as a diagnostic reagent.

Example 4 Anti-PD-1 Antibodies Sequences and Subsequent Humanization

Cloning of Immunoglobulin cDNAs

Using degenerate primer PCR-based methods, the DNA sequences encodingthe variable regions of the mouse antibodies expressed by hybridomashPD-1.08A and hPD-1.09A were determined. Briefly, gene specific cDNAsfor the heavy and light chains were generated using the iScript SelectcDNA synthesis kit (Biorad # 1708896) according to the manufacturer'sinstructions. PCR primers used were based on the Ig-primer set (Novagen# 69831-3). Degenerate PCR reactions were carried out using Taqpolymerase according to the Novagen primer set protocol. PCR productswere analyzed by agarose gel electrophoresis. The expected amplicon sizefor both the heavy and light chain variable region is about 500 basepairs. Two μl of Taq-amplified PCR product from reactions which yieldedan appropriate band were cloned into the pCR4 TOPO vector (Invitrogen#K4595-40) and transformed into DH5-alpha E. coli as directed by themanufacturer. Clones were screened by colony PCR using universal M13forward and reverse primers and two to three clones from each reactionwere chosen for DNA sequencing analysis.

Clones were sequenced in both directions using universal primers M13forward, M13 reverse, T3 and T7. Results of each sequencing reaction foreach clone were analyzed using Seqman. Consensus sequences were searchedagainst databases of germline and rearranged Ig Variable regionsequences using NCBI Ig-Blast(http://www.ncbi.nlm.nih.gov/projects/igblast/). Blast results forhPD-1.08A identified a productively (in-frame) rearranged heavy chainwith no stop codons introduced. Light chain clones were identified whichencode two different sequences; one is a productively (in-frame)rearranged light chain with no stop codons introduced, the other is anon-productively rearranged sequence containing a frame-shift leading toa stop codon in the FR4 region. The non-productive sterile transcriptobserved likely originates from the myeloma fusion partner (Carroll W.L. et al., Mol. Immunol. 25:991-995 (1988) and was ruled out.

Blast results for hPD-1.09A identified productively (in-frame)rearranged heavy and light chains with no stop codons introduced. Theamino acid sequences of the expressed proteins were been confirmed bymass spectrometry. The sequences are disclosed in the attached SequenceListing and listed in table IV.

TABLE IV Sequence ID numbers for murine anti- human PD-1 antibodies ofthis invention SEQ ID NO: Description 1 hPD-1.08A heavy chain variableregion (DNA) 2 hPD-1.08A light chain variable region (DNA) 3 hPD-1.09Aheavy chain variable region (DNA) 4 hPD-1.09A light chain variableregion (DNA) 5 hPD-1.08A heavy chain variable region (AA) 6 hPD-1.08Alight chain variable region (AA) 7 hPD-1.09A heavy chain variable region(AA) 8 hPD-1.09A light chain variable region (AA) 9 hPD-1.08A lightchain CDR1 (AA) 10 hPD-1.08A light chain CDR2 (AA) 11 hPD-1.08A lightchain CDR3 (AA) 12 hPD-1.08A heavy chain CDR1 (AA) 13 hPD-1.08A heavychain CDR2 (AA) 14 hPD-1.08A heavy chain CDR3 (AA) 15 hPD-1.09A lightchain CDR1 (AA) 16 hPD-1.09A light chain CDR2 (AA) 17 hPD-1.09A lightchain CDR3 (AA) 18 hPD-1.09A heavy chain CDR1 (AA) 19 hPD-1.09A heavychain CDR2 (AA) 20 hPD-1.09A heavy chain CDR3 (AA) 21 109A-H heavy chainvariable region (DNA) 22 Codon optimized 109A-H heavy chain variableregion (DNA) 23 Codon optimized 409A-H heavy chain full length (DNA) 24K09A-L-11 light chain variable region (DNA) 25 K09A-L-16 light chainvariable region (DNA) 26 K09A-L-17 light chain variable region (DNA) 27Codon optimized K09A-L-11 light chain variable region (DNA) 28 Codonoptimized K09A-L-16 light chain variable region (DNA) 29 Codon optimizedK09A-L-17 light chain variable region (DNA) 30 109A-H heavy chainvariable region (AA) 31 409A-H heavy chain full length (AA) 32 K09A-L-11light chain variable region (AA) 33 K09A-L-16 light chain variableregion (AA) 34 K09A-L-17 light chain variable region (AA) 35 109A-Hheavy chain full length (AA) 36 K09A-L-11 light chain full length (AA)37 K09A-L-16 light chain full length (AA) 38 K09A-L-17 light chain fulllength (AA)

CDR and framework regions are annotated according to Kabat E. A., etal., 1991, Sequences of proteins of Immunological interest, In: NIHPublication No. 91-3242, US Department of Health and Human Services,Bethesda, Md.

Construction and Expression of Chimeric c109A Antibody

Chimeric light and heavy chains were constructed by linking thePCR-cloned cDNAs of mouse hPD-1.09A VL and VH regions to human kappa andIgG1 constant regions, respecitvely. The 5′ and 3′ ends of the mousecDNA sequences were modified using PCR primers designed to add asuitable leader sequence to each chain, and restriction sites to enablecloning into existing recombinant antibody expression vectors.

COS-7 cells (0.7 mL at 10⁷/mL) were electroporated with 10 μg of each ofthe chimeric heavy and light chain expression plasmids. These cells werethen cultured in 8 mL growth medium for three days. A sandwich ELISA wasused to measure the antibody concentrations in the supernatants from theCOS-7 transfections. This showed that the transfected COS-7 cellssecreted about 295 ng/mL of the chimeric IgG₁-kappa antibody in threeseparate transfections.

Binding of the chimeric antibody produced by the transfected COS-7 cellswas measured using PD-1 binding ELISA and CELISA (see Example 2) and wasshown to bind to PD-1 with comparable affinity to that of the murineantibody.

Humanized Antibody Design

The hPD-1.09A antibody was humanized by MRCT (Cambridge UK) using CDRgrafting technology (see, e.g., U.S. Pat. No. 5,225,539). Briefly, thevariable chain sequences of the murine antibody hPD-1.09A were comparedto those available in the Research Collaboratory for StructuralBioinformatics (RCSB) protein databank. A homology model of hPD-1.09Awas generated based on the nearest VH and VK structures. Human sequenceswith highest identity to hPD-1.09A were identified and analyzed. (Footeand Winter, J. Mol. Biol. 224:487-499 (1992); Morea V. et al., Methods20:267-279 (2000); Chothia C. et al., J. Mol. Biol. 186:651-663 (1985).)The most appropriate human frameworks on which to build the CDR graftedheavy and light chains were identified.

For the heavy chain, the framework encoded by genbank accession #AB063829 was determined to be the most appropriate. Analysis of thehPD-1.09A VK sequence shows that its CDR1 length (15 residues) is notfound in any human VK. For this reason, frameworks of three differentCDR1 lengths (11, 16 and 17 residues) were analyzed in order to testwhich CDR1 length would reproduce the behavior of hPD-1.09A VK. Thehuman VK sequences with highest identity to hPD-1.09A VK at selectedresidues important in the structure and with CDR1 lengths 11, 16 and 17were identified. The framework of genbank accession # M29469 wasselected on which to base K109A-L-11. The framework from genbankaccession # AB064135 was selected on which to base K09A-L-16 and theframework from genbank accession # X72431 was chosen on which to baseK09A-L-17.

Straight grafts were performed to generate expression constructs foreach chain. The DNA and protein sequences of 109A-H, K09A-L-11,K09A-L-16 and K09A-L-17 are disclosed in the attached Sequence Listing(Table IV).

An IgG4 version of the humanized h109A antibody was produced, with thestabilizing Adair mutation (Angal S. et al., Mol. Immuol. 30:105-108(1993)), where serine 241 (Kabat numbering) is converted to proline.This sequence is disclosed in SEQ ID NOS: 23 and 31.

Example 5 Binding Characteristics and Functional Properties of Humanizedanti-PD-1 Antibodies Production and Purification

Humanized antibodies h409A11, h409A16 and h409A17 were produced bytransient transfection of CHO-S cells. Cells were grown in CD-CHO(Gibco) and C5467 media (Sigma) for 8 days in shaker flasks. Antibodieswere purified from cell supernatants by Protein A chromatography,washed, eluted using 1 M acetic acid and neutralized using 3 M Tris.Finally, the buffer was exchanged for 100 mM acetic acid which had beenadjusted to pH 5.5 with 1 M Tris base.

Binding and Kinetic Analysis

Protein-based and cell-based ELISAs to determine apparent bindingaffinities (reported as EC50 values) were performed as described inExample 2. The humanized anti-PD-1 antibodies each bound to PD-1/Fc andcellularly expressed PD-1 with comparable EC50 values to the murineparent antibody (Table V).

Kinetic binding characteristics of the antibodies were also performedusing bio-light interferometry as described in Example 2 (FIG. 6). Twoof the humanized antibodies, h409A11 and h409A16, bound considerablymore tightly than any other mAb tested using this assay, with K_(D)determined to be 29 and 27 μM for h409A11 and h409A16, respectively(Table V). Compared to the other anti-PD-1 antibodies tested, theincreased affinity is mainly due to a slower off-rate. Similar to themurine parental antibodies, the humanized anti-PD-1 antibodies h409A11,h409A16 demonstrated binding to cynomolgous PD-1 with K_(D) determinedto be below 120 pM.

Ligand Blockade

The ability of the humanized antibodies to block the binding of PD-L1and PD-L2 to PD-1 was measured using a homogeneous competition assay anddetection using an FMAT competition assay as described in Example 2.

Binding of both hPD-L1/Fc and hPD-L2/Fc to CHO-hPD-1 cells can becompletely inhibited in a dose-dependent fashion by any of the humanizedantibodies tested. Calculated IC₅₀ data are summarized in Table V.Similarly to the parent murine antibody hPD-1.09A, each of the humanizedmAbs, h409A11, h409A16 and h409A17 inhibited PD-L1 and PD-L2 bindingwith IC₅₀ values below 1 nM. Similar to the murine parental antibodies,the humanized anti-PD-1 antibodies h409A11, h409A16 and h409A17demonstrated inhibition of ligand binding to cynomolgous PD-1 withcalculated IC₅₀ values under about 1 nM.

TABLE V Binding characteristics of humanized anti-hPD-1 antibodies ofthe invention Binding Analysis Ligand Blockade Kinetic Analysis ELISACELISA FMAT Fortebio Octet EC50 (pM) EC50 (pM) IC50 (pM) k_(assoc)k_(dissoc) K_(D) mAb hPD-1/Fc hPD-1/CHO PD-L1 PD-L2 1/s 1/Ms M h409A1176 62 625 695 1.04E+06 3.05E−05 2.93E−11 h409A16 90 63 696 810 9.97E+052.72E−05 2.73E−11 h409A17 88 83 818 463 1.00E+06 1.91E−04 1.91E−10Human T Cell Response to SEB is Enhanced by Humanized mABS

Humanized anti-PD-1 antibodies were tested for their capacity to enhanceT cell activity in vitro using blood cells from healthy volunteers asdescribed in Example 3. Supernatants were collected after 4 days ofculture and the amount of IL-2 produced was quantified using ELISA Thehumanized PD-1 antibodies demonstrated the capacity to increase IL-2production stimulated by SEB (FIG. 7). Additionally, the humanized PD-1antibodies increased SEB induced IL-2 production in cancer patientblood, similar to what is described in Example 3.

In summary, the humanized mAbs h409A11, h409A16, and h409A17 retainedall functional activity during the humanization process. The h409A11 andh409A16 mAbs fully retained the affinity of the mouse parental antibodyhPD109A upon humanization.

1. An isolated antibody or antibody fragment which binds to human PD-1comprising: a. at least one CDR selected from the group consisting ofSEQ ID NOs: 9, 10, 11, 15, 16 and 17, or a variant of any said sequence;and/or b. at least one a CDR selected from the group consisting of SEQID NOs: 12, 13, 14, 18, 19 and 20, or a variant of any said sequence. 2.The antibody or antibody fragment of claim 1, comprising: a. light chainCDRs SEQ ID NOs: 9, 10 and 11, or variants of any said sequences; andheavy chain CDRs SEQ ID NOs: 12, 13 and 14, or variants of any saidsequences; or b. light chain CDRs SEQ ID NOs: 15, 16 and 17 or variantsof any said sequences; and heavy chain CDRs SEQ ID NOs: 18, 19 and 20 orvariants of any said sequences.
 3. The antibody or antibody fragment ofclaim 2, comprising: a. a heavy chain variable region comprising anamino acid sequence selected from the group consisting of i. SEQ ID NO:5 or a variant thereof; ii SEQ ID NO: 7 or a variant thereof; iii. aminoacid residues 20 to 139 of SEQ ID NO: 30 or a variant thereof; and iv.an amino acid sequence having at least 90% homology to amino acidresidues 20 to 139 of SEQ ID NO: 30; and further comprising b. a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of: i. SEQ ID NO: 6 or a variant thereof; ii. SEQID NO: 8 or a variant thereof; iii. amino acid residues 20 to 130 of SEQID NO: 32 or a variant thereof; iv. amino acid residues 20 to 130 of SEQID NO: 33 or a variant thereof; v. amino acid residues 20 to 130 of SEQID NO: 34 or a variant thereof; and vi. an amino acid sequence having atleast 90% homology to amino acid residues 20 to 130 of SEQ ID NO: 32, 33or
 34. 4. The antibody of claim 1, comprising: a. a heavy chaincomprising an amino acid sequence selected from the group consisting of:i. amino acid residues 20 to 466 of SEQ ID NO: 31 or a variant thereof,and ii. amino acid residues 20 to 469 of SEQ ID NO: 35 or a variantthereof; and b. a light chain comprising an amino acid sequence selectedfrom the group consisting of: i. amino acid residues 20 to 237 of SEQ IDNO: 36 or a variant thereof; ii. amino acid residues 20 to 237 of SEQ IDNO:37 or a variant thereof, and iii. amino acid residues 20 to 237 ofSEQ ID NO: 38 or a variant thereof.
 5. The antibody or antibody fragmentof claim 1, wherein any of said variant(s) may comprise up to threeconservatively modified amino acid substitutions.
 6. The antibody ofclaim 1 further comprising: a human heavy chain constant region or avariant thereof, wherein the variant comprises up to 20 conservativelymodified amino acid substitutions; and/or b. a human light chainconstant region or a variant thereof, wherein the variant comprises upto 20 conservatively modified amino acid substitutions.
 7. The antibodyof claim 6, wherein the human heavy chain constant region comprises a γ4or γ1 human heavy chain constant region or a variant thereof, whereinthe variant comprises up to 20 conservatively modified amino acidsubstitutions.
 8. The antibody or antibody fragment of claim 1, whereinthe antibody or antibody fragment: a. binds human PD-1 with a K_(D) ofabout 100 pM or lower; b. binds human PD-1 with a K_(D) of about 30 pMor lower; c. binds to human PD-1 with about the same K_(D) as anantibody having a heavy chain comprising the amino acid sequence of SEQID NO: 31 and a light chain comprising the amino acid sequence of SEQ IDNO: 32; d. binds to human PD-1 with about the same K_(D) as an antibodyhaving a heavy chain comprising the amino acid sequence of SEQ ID NO: 31and a light chain comprising the amino acid sequence of SEQ ID NO: 33;e. binds to human PD-1 with a k_(assoc) of about 7.5×10-5 l/M·s orfaster; f. binds to human PD-1 with a k_(assoc) of about 1×106 l/M·s orfaster; g. binds to human PD-1 with a k_(dissoc) of about 2×10⁻⁵ l/s orslower; h. binds to human PD-1 with a k_(dissoc) of about 2.7×10⁻⁵ l/sor slower; i. binds to human PD-1 with a k_(dissoc) of about 3×10⁻⁵ l/sor slower; or j. blocks binding of human PD-L1 or human PD-L2 to humanPD-1 with an IC₅₀ of about 1 nM or lower.
 9. An isolated antibody orantibody fragment which competes for a binding epitope on PD-1 with theantibody or antibody fragment of claim 1, and has a one of the followingcharacteristics: a. binds human PD-1 with a K_(D)) of about 100 pM orlower; b. binds human PD-1 with a K_(D) of about 30 pM or lower; c.binds to human PD-1 with about the same K_(D) as an antibody having aheavy chain comprising the amino acid sequence of SEQ ID NO: 31 and alight chain comprising the amino acid sequence of SEQ ID NO: 32; d.binds to human PD-1 with about the same K_(D) as an antibody having aheavy chain comprising the amino acid sequence of SEQ ID NO: 31 and alight chain comprising the amino acid sequence of SEQ ID NO: 33; e.binds to human PD-1 with a k_(assoc) of about 7.5×105 l/M·s or faster;f. binds to human PD-1 with a k_(assoc) of about 1×106 l/M·s or faster;g. binds to human PD-1 with a k_(dissoc) of about 2×10-5 l/s or slower;h. binds to human PD-1 with a k_(dissoc) of about 2.7×10-5 l/s orslower; i. binds to human PD-1 with a k_(dissoc) of about 3×10-5 l/s orslower; or j. blocks binding of human PD-L1 or human PD-L2 to human PD-1with an IC₅₀ of about 1 nM or lower.
 10. The antibody or antibodyfragment of claim 1, wherein the antibody or antibody fragment is: a. achimeric antibody or a fragment thereof; b. a human antibody or afragment thereof; c. a humanized antibody or a fragment thereof; or d.an antibody fragment selected from the group consisting of Fab, Fab′,Fab′-SH, Fv, scFv. F(ab′)₂, and a diabody.
 11. The antibody or antibodyfragment of claim 1, wherein the antibody or antibody fragment increasesactivation of T cells.
 12. An isolated polynucleotide encoding theantibody or antibody fragment of claim
 1. 13. An expression vectorcomprising the isolated polynucleotide of claim
 12. 14. A host cellcomprising the expression vector of claim
 13. 15. A method of producingan antibody or antibody fragment comprising: a. culturing the host cellof claim 14 in culture medium under conditions wherein the nucleic acidsequence is expressed, thereby producing polypeptides comprising thelight and heavy chain variable regions; and b. recovering thepolypeptides from the host cell or culture medium.
 16. A compositioncomprising the antibody or antibody fragment of claim 1, in combinationwith a pharmaceutically acceptable carrier or diluent.
 17. A method ofincreasing the activity of an immune cell, comprising contacting theimmune cell with the antibody or antibody fragment of claim
 1. 18. Amethod of increasing the activity of an immune cell, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an antibody or antibody fragment of claim
 1. 19. The method ofclaim 18, wherein said method is used for: a. the treatment of cancer;b. the treatment of an infection or infectious disease; or c. as avaccine adjuvant.
 20. The method of claim 18, further comprisingmeasuring 1 cell activation ex vivo in a sample derived from thesubject, wherein an increase in T cell activity indicates that thetreatment should be continued or predicts the likelihood that thetreatment will be successful.
 21. The method of claim 20, wherein theincrease in T cell activity is determined by: a. measuring SEB inducedproduction of one or more cytokines selected from the group consistingof: IL-2, TNFα, IL-17, IFNγ, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13;or b. measuring TT induced production of a cytokine selected from thegroup consisting of: IL-2, TNFα, IL-17, IFNγGM-CSF, RANTES, IL-6, IL-8,IL-5 and IL-13.
 22. (canceled)
 23. (canceled)